Augmented vasoconstrictor reactivity is thought to play an important role in the development of chronic hypoxia (CH)-induced neonatal pulmonary hypertension. However, whether this response to CH results from pulmonary endothelial dysfunction and reduced nitric oxide (NO)-mediated vasodilation is not well understood. We hypothesized that neonatal CH enhances basal tone and pulmonary vasoconstrictor sensitivity by limiting NO-dependent pulmonary vasodilation. To test this hypothesis, we assessed the effects of the NO synthase (NOS) inhibitor Nω-nitro-l-arginine (l-NNA) on baseline pulmonary vascular resistance (PVR) and vasoconstrictor sensitivity to the thromboxane mimetic U-46619 in saline -perfused lungs (in situ) from 2-wk-old control and CH (12-day exposure, 0.5 atm) Sprague-Dawley rats. Basal tone was defined as that reversed by exogenous NO (spermine NONOate). CH neonates displayed elevated right ventricular systolic pressure (in vivo) and right ventricular hypertrophy, indicative of pulmonary hypertension. Perfused lungs from CH rats demonstrated greater baseline PVR, basal tone, and U-46619-mediated vasoconstriction compared with control rats in the absence of l-NNA. l-NNA markedly increased baseline PVR and reactivity to U-46619 in lungs from CH neonates, further augmenting vasoconstrictor sensitivity compared with control lungs. Exposure to CH also enhanced NO-dependent vasodilation to arginine vasopressin, pulmonary expression of NOS III [endothelial NOS (eNOS)], and eNOS phosphorylation at activation residue Ser1177. However, CH did not alter lung nitrotyrosine levels, a posttranslational modification reflecting [Formula: see text] scavenging of NO. We conclude that, in contrast to our hypothesis, enhanced basal tone and agonist-induced vasoconstriction after neonatal CH is limited by increased NO-dependent pulmonary vasodilation resulting from greater eNOS expression and phosphorylation at activation residue Ser1177. NEW & NOTEWORTHY This research is the first to demonstrate enhanced nitric oxide-dependent vasodilation that limits increased vasoconstrictor reactivity in neonatal pulmonary hypertension. These results suggest that augmented vasoconstriction in this setting reflects changes in smooth muscle reactivity rather than a reduction in nitric oxide-dependent pulmonary vasodilation.

Rats fed a high fat diet develop increased adiposity and oxidative stress leading to impaired vasodilation. The purpose of the present study was to examine the effects of high fat-induced increases in adiposity and oxidative stress on vasoconstrictor reactivity of isolated mesenteric arteries. We hypothesized that rats with more adiposity would develop oxidative stress-potentiated increases in iNOS-derived nitric oxide leading to diminished vasoconstriction. Male Sprague-Dawley rats were fed either a control (Chow) or high fat diet for 6 weeks. The roles of oxidative stress and iNOS in the impaired vasoconstrictor responses to endothelin-1 were characterized in small mesenteric arteries. Rats fed the HFD developed significantly more adiposity compared to Chow rats. Plasma levels of nitric oxide and the inflammatory factor tumor necrosis factor α were significantly higher in high fat fed rats compared to Chow rats (nitric oxide: 95.36±19.3 vs. 38.96±6.7 μM; tumor necrosis factor α: 598±111.4 vs. 292±71.8 pg/ml, respectively). Despite exhibiting elevated systolic blood pressure compared to Chow rats (153.5±2.4 vs. 137.5±2.7 mm Hg), endothelin-1 mediated vasoconstriction was impaired in isolated mesenteric arteries from high fat fed rats but was normalized by individual or combined inhibition of nitric oxide synthase, iNOS, or oxidative stress. Therefore, oxidative stress and iNOS are involved in the attenuation of endothelin-1 mediated vasoconstriction observed in isolated mesenteric arteries from high fat fed rats.

Effects of chronic hypoxia (CH) on store- and receptor-operated Ca2+ entry (SOCE, ROCE) in pulmonary vascular smooth muscle (VSM) are controversial, although whether genetic variation explains such discrepancies in commonly studied rat strains is unclear. Since protein kinase C (PKC) can inhibit Ca2+ permeable nonselective cation channels, we hypothesized that CH differentially alters PKC-dependent inhibition of SOCE and ROCE in pulmonary VSM from Sprague-Dawley and Wistar rats. To test this hypothesis, we examined SOCE and endothelin-1 (ET-1)-induced ROCE in endothelium-disrupted, pressurized pulmonary arteries from control and CH Sprague-Dawley and Wistar rats. Basal VSM Ca2+ was elevated in CH Wistar, but not Sprague-Dawley, rats. Further, CH attenuated SOCE in VSM from Sprague-Dawley rats, while augmenting this response in Wistar rats. CH reduced ROCE in arteries from both strains. PKC inhibition restored SOCE in CH Sprague-Dawley arteries to control levels, while having no effect on SOCE in Wistar arteries or on ROCE in either strain. We conclude that effects of CH on pulmonary VSM SOCE are strain dependent, whereas inhibitory effects of CH on ROCE are strain independent. Further, PKC inhibits SOCE following CH in Sprague-Dawley, but not Wistar, rats but does not contribute to ET-1-induced ROCE in either strain.

Objective: Exposure to chronic hypoxia (CH) results in a persistent endothelium-dependent vascular smooth muscle hyperpolarization that diminishes vasoconstrictor reactivity. Experiments were performed to test the hypothesis that products of both cytochrome P450 epoxygenase (CYP) and heme oxygenase (HO) are required for the persistent diminished myogenic reactivity following CH.Methods: The authors examined myogenic responses of mesenteric arteries isolated from control and CH (48 h; P-B = 380 mmHg) rats in the presence of a HO inhibitor (zinc protoporphyrin IX; ZnPPIX) or combined HO and CYP epoxygenase inhibition (sulfaphenazole). Arteries were isolated and cannulated and the vascular smooth muscle was loaded with the Ca2+ indicator Fura-2.Results: Control vessels maintained their internal diameter in response to step increases in intraluminal pressure, whereas arteries from CH animals passively distended. ZnPPIX augmented myogenic reactivity and [Ca2+] in arteries from CH animals. Combined administration of sulfaphenazole and ZnPPIX did not have an additional effect compared to ZnPPIX alone. Myogenic reactivity in control vessels was not altered by ZnPPIX or ZnPPIX + sulfaphenazole.Conclusions: HO appears to play a role in regulating myogenic reactivity following CH. Furthermore, these data suggest that products of HO and CYP are both required for the observed attenuation in vasoreactivity following CH.

Recent evidence from our laboratory and others indicates that chronic hypoxia (CH) augments agonist-induced RhoA and Rho kinase (ROK) activity, increases ROK expression, and enhances ROK-dependent myofilament Ca2+ sensitivity in pulmonary vascular smooth muscle (VSM). The RhoA/ROK signaling pathway has been further implicated in mediating pulmonary VSM contraction to endothelin-1 (ET-1), an endothelium-derived vasoconstrictor peptide that contributes to the development of CH-induced pulmonary hypertension. We therefore hypothesized that CH augments ET-1-mediated Ca2+ sensitization in pulmonary VSM. To test this hypothesis, we assessed vasoconstrictor responses to ET-1 (10−10–10−7 M) in endothelium-denuded, pressurized pulmonary arteries (151 ± 12 μm inner diameter) from control and CH (4 wk at PB = 380 mmHg) rats. Arteries were permeabilized to Ca2+ with ionomycin (3 μM), superfused with a physiological saline solution containing 300 nM Ca2+, and loaded with fura-2 AM to verify that VSM intracellular free Ca2+ concentration was maintained constant during ET-1-induced vasoconstriction. In agreement with our hypothesis, vasoconstriction to ET-1 (10−9–10−7 M) was markedly enhanced in CH vs. control arteries. We conclude that CH increases ET-1-induced myofilament Ca2+ sensitivity in pulmonary VSM. (Supported by NIH grants HL-07736, HL-77876, HL-58124 and HL-63207)

Chronic hypoxia (CH) elicits blunted vasoconstrictor reactivity in the mesenteric vascular bed that is likely due to tonic endothelium-dependent vascular smooth muscle (VSM) hyperpolarization. CH-induced VSM hyperpolarization is not reversed by nitric oxide synthase (NOS) or cyclooxygenase (COX) inhibition, suggesting a role of an endothelium-derived hyperpolarizing factor (EDHF). Stimulation of endothelial calcium-activated K+ channels by agonists such as acetylcholine (ACh) causes an efflux of K+ and cell hyperpolarization. We hypothesize that this hyperpolarization passively spreads to the underlying VSM via myoendothelial gap junctions differentially in arteries from CH rats compared to controls. Mesenteric arterioles from control and CH rats were mounted in a pressure myograph and vessel inner diameter recorded. ACh (1 μM) -induced dilations of arteries partially preconstricted with phenylephrine (PE) were unaffected by combined COX and NOS blockade (% reversal control: 94.97 ± 1.33 (n=6), CH: 94.75 ± 0.52 (n=5)), supportive of an EDHF-mediated response. In contrast, EDHF-mediated dilation was significantly (p<0.05) attenuated by treatment with a high K+(125mM) superfusate in both groups (% reversal control: 27.36 ± 4.70 (n=5), CH: 34.08 ± 4.08 (n=5)). Also, EDHF-mediated dilation was significantly (p<0.05) reduced by treatment with the gap junction inhibitor carbenoxolone (100 μM) in both groups (% reversal control: 33.98 ± 9.94 (n=5), CH: 38.03 ± 17.78 (n=6)). These results suggest that K+ channels and gap junctions are involved in ACh-induced responses, but that CH does not alter the nature of EDHF-mediated dilation.

We previously observed that in mesenteric arteries from intermittent hypoxia-induced (IH) hypertensive rats endothelin-1 (ET-1) constriction is augmented compared to constriction in Sham arteries. Furthermore, in Sham arteries, ET-1 constriction was accompanied by increases in vessel wall [Ca2+] but in IH arteries, constriction occurred without increases in [Ca2+]. Thus IH appears to augment ET-1 constriction by increasing vascular smooth muscle Ca-sensitizing pathways. We hypothesized that IH increased ET-1 activation of either protein kinase C (PKC) or Rho associated kinase (ROK) to augment Ca-sensitivity. We observed that ROK inhibition (Y-27632, 3 μM) attenuated ET-1 constriction slightly more in IH arteries than in Sham arteries (IH 80±3, Sham 89±4 % control ET-1 constriction). However, PKC inhibition (GF-109203x, 3 μM) greatly attenuated ET-1 mediated constriction in IH arteries but did not affect ET-1 constriction in Sham arteries (IH 50±3*, Sham 98±4 % control ET-1 constriction, *P<0.05). This suggests that IH greatly increases ET-1 activation of PKC-mediated constriction and slightly increases ET-1 activation of ROK-dependent constriction. Western analysis suggested that there is a tendency for PKC-α expression to be increased in IH mesenteric arteries compared to Sham but the difference was not significant (P = 0.33). Therefore augmented ET-1 vasoconstriction in IH arteries is mediated almost exclusively through Ca-sensitization with a large contribution by PKC activation that appears to be independent of IH-induced increases PKC-α expression. (Supported by AHA Established Investigator award and EPA RD-83186001, NLK)

We have recently demonstrated that store-operated and UTP-induced receptor-operated Ca2+ entry (ROCE) are attenuated in pulmonary vascular smooth muscle following chronic hypoxia (CH). However, it is not clear whether this inhibitory effect of CH on ROCE represents a generalized response to receptor-mediated vasoconstrictor agonists. We hypothesized that CH similarly diminishes ROCE in response to endothelin-1 (ET-1), an endothelium-derived vasoconstrictor peptide that contributes to the development of CH-induced pulmonary hypertension. To test this hypothesis, we examined ET-1-induced ROCE in isolated, endothelium-denuded and pressurized pulmonary arteries (167 ¡Ó 18 £gm inner diameter) from control and CH (4 wk at 0.5 atm) rats. Arteries were loaded with fura-2 AM to continuously monitor VSM [Ca2+]i. To isolate ROCE, we inhibited L-type voltage-operated Ca2+ channels with diltiazem (50 ƒÝM), and depleted intracellular Ca2+ stores with cyclopiazonic acid (10 ƒÝM) to induce influx of Ca2+ through store-operated channels. The further increase in [Ca2+]i observed upon stimulation with ET-1 (10-10-10-7 M) was attributed to ROCE. We found that the change in VSM [Ca2+]i to 10-8-10-7 M ET-1 was significantly attenuated (p<0.05) in arteries from CH rats compared to controls. We conclude that CH inhibits ET-1-mediated ROCE in pulmonary VSM and may be reflective of a generalized inhibition of this pathway in the setting of CH. (Supported by NIH grants HL-07736, HL-77876, HL-58124 and HL-63207)

Chronic hypoxia (CH) results in an endothelium-dependent blunting of myogenic responsiveness in isolated rat skeletal muscle arteries. We hypothesized that this attenuated myogenic responsiveness is due to an alteration of large conductance Ca2+-activated K+ (BKCa) channel activity in the endothelial cell of CH arteries. Third order branches of the caudal femoral artery (150–200 μm) from normoxic control rats and rats exposed to hypoxia (PB = 380 mmHg for 48 hours), were isolated, cannulated and pressurized. Vessel inner diameter was recorded at intraluminal pressures of 20–200 mmHg under normoxic conditions. Blunted myogenic responsiveness of CH arteries persisted in the presence of N-Nitro-L-arginine (L-NNA) (100 μM) and indomethacin (10 μM) and after further treatment with the luminally administered small and intermediate conductance potassium channel blockers, apamin (100 nM) and TRAM 34 (1 μM), respectively. However, myogenic responsiveness of CH arteries was restored to control levels by luminal administration of either a combination of charybdotoxin (100 nM) and apamin (100 nM) (n=5, both groups) or iberiotoxin (100 nM) alone (n=5, both groups), in the continued presence of L-NNA and indomethacin. These latter treatments did not affect myogenic reactivity in arteries from control rats. We conclude that endothelial cell BKCa channel activity is increased in CH arteries resulting in hyperpolarization that is conducted to the underlying vascular smooth muscle thereby attenuating myogenic responsiveness.

We have recently demonstrated that chronic hypoxia (CH) attenuates nitric oxide (NO)-mediated decreases in pulmonary vascular smooth muscle (VSM) intracellular free calcium concentration ([Ca2+]i) and promotes NO-dependent VSM Ca2+ desensitization. The objective of the current study was to identify potential mechanisms by which CH interferes with regulation of [Ca2+]i by NO. We hypothesized that CH impairs NO-mediated inhibition of store-operated (capacitative) Ca2+ entry (SOCE) or receptor-operated Ca2+ entry (ROCE) in pulmonary VSM. To test this hypothesis, we examined effects of the NO donor, spermine NONOate, on SOCE resulting from depletion of intracellular Ca2+ stores with cyclopiazonic acid, and on UTP-induced ROCE in isolated, endothelium-denuded, pressurized pulmonary arteries (213 +/- 8 microm inner diameter) from control and CH (4 wk at 0.5 atm) rats. Arteries were loaded with fura-2 AM to continuously monitor VSM [Ca2+]i. We found that the change in [Ca2+]i associated with SOCE and ROCE was significantly reduced in vessels from CH animals. Furthermore, spermine NONOate diminished SOCE and ROCE in vessels from control, but not CH animals. We conclude that NO-mediated inhibition of SOCE and ROCE is impaired after CH-induced pulmonary hypertension.

Bilirubin is a potent antioxidant that acts in a cytoprotective manner to diminish levels of reactive oxygen species (ROS). Heme oxygenase (HO) produces bilirubin through the breakdown of heme into biliverdin, which is subsequently converted to bilirubin by biliverdin reductase. These enzymatic pathways are present in many tissues including the vascular endothelium. We hypothesized that inhibition of HO would result in increased ROS levels within the vasculature. To quantify ROS, isolated mesenteric resistance arterioles from Sprague-Dawley rats were superfused with the fluorescent indicator DCF. Specificity of DCF fluorescence as a measure of ROS was verified using Tiron and catalase, scavengers of superoxide and hydrogen peroxide, respectively. Addition of these compounds to the superfusate significantly decreased ROS levels in mesenteric arterioles from 109.2 ± 7.36 arbitrary units (AU), to 24.37 ± 7.22 AU. Catalase alone decreased ROS levels to a similar extent as combined Tiron and catalase suggesting that the major ROS formed in these vessels is hydrogen peroxide. Supportive of our hypothesis, superfusion with the HO inhibitor zinc protoporphyrin IX (ZnPPIX; 0.5μM) resulted in augmented DCF fluorescence (250.2 ± 25.19 AU). These data suggest that the HO system is an important regulator of ROS levels within the resistance vasculature.

Chronic obstructive pulmonary diseases, as well as prolonged residence at high altitude, can result in generalized airway hypoxia, eliciting an increase in pulmonary vascular resistance. We hypothesized that a portion of the elevated pulmonary vascular resistance following chronic hypoxia (CH) is due to the development of myogenic tone. Isolated, pressurized small pulmonary arteries from control (barometric pressure congruent with 630 Torr) and CH (4 wk, barometric pressure = 380 Torr) rats were loaded with fura 2-AM and perfused with warm (37 degrees C), aerated (21% O(2)-6% CO(2)-balance N(2)) physiological saline solution. Vascular smooth muscle (VSM) intracellular Ca(2+) concentration ([Ca(2+)](i)) and diameter responses to increasing intraluminal pressure were determined. Diameter and VSM cell [Ca(2+)](i) responses to KCl were also determined. In a separate set of experiments, VSM cell membrane potential responses to increasing luminal pressure were determined in arteries from control and CH rats. VSM cell membrane potential in arteries from CH animals was depolarized relative to control at each pressure step. VSM cells from both groups exhibited a further depolarization in response to step increases in intraluminal pressure. However, arteries from both control and CH rats distended passively to increasing intraluminal pressure, and VSM cell [Ca(2+)](i) was not affected. KCl elicited a dose-dependent vasoconstriction that was nearly identical between control and CH groups. Whereas KCl administration resulted in a dose-dependent increase in VSM cell [Ca(2+)](i) in arteries taken from control animals, this stimulus elicited only a slight increase in VSM cell [Ca(2+)](i) in arteries from CH animals. We conclude that the pulmonary circulation of the rat does not demonstrate pressure-induced vasoconstriction.

We reported previously that simulating sleep apnea in rats by exposing them 7 hours per day to intermittent hypoxia/hypercapnia (IH) elevates plasma endothelin-1 and causes hypertension, which is reversed by an endothelin-1 antagonist. We hypothesized that in this model of sleep apnea-induced hypertension, vascular sensitivity to endothelin-1 is increased in combination with the elevated plasma endothelin-1 to cause the endothelin-1-dependent hypertension. In small mesenteric arteries with endothelial function disabled by passing air through the lumen, diameter and vessel wall [Ca2+] were recorded simultaneously. IH arteries demonstrated increased constrictor sensitivity to endothelin-1 (percentage max constriction 100 +/- 0% IH versus 80 +/- 10% Sham; P<0.05). This was accompanied by increased calcium sensitivity of IH arteries. In contrast, constrictor sensitivity and increases in vessel wall [Ca2+] to KCl and phenylephrine were not different between IH and Sham arteries. We have shown previously that endothelin-1 constriction in mesenteric arteries is mediated by endothelin A receptors. In the current study, the selective increase in endothelin-1 constriction in IH resistance arteries was accompanied by increased expression of endothelin A receptor expression (densitometry units 271 +/- 23 IH versus 158 +/- 25 Sham; P<0.05). Thus, IH hypertension appears to cause alterations in signaling components unique to endothelin-1 at the receptor level and in postreceptor signaling that increases calcium sensitivity during endothelin A activation. Future studies will determine the specific changes in vascular smooth muscle signaling in IH hypertension causing this augmented contractile phenotype.